Regeneration of sensory hair cells in the mature cochlea is a major therapeutic challenge. Atoh1, a transcription factor important for hair cell development, can induce hair cell formation from surrounding supporting cells in postnatal murine cochleae. However, hair cells regenerated by Atoh1 are too few, are incompletely mature, and most importantly, exhibit age-dependent decline in the postnatal cochlea. It remains unclear which molecules and signaling pathways modulate Atoh1-mediated conversion of supporting cells to hair cells in the mature cochlea in vivo. Our preliminary results demonstrate that genetic manipulation of additional factors, in conjunction with Atoh1 activation, in supporting cells can convert them to hair cells in the mature cochlea in vivo. Remarkably, we also showed that one of the three factors by itself can convert supporting cells to hair cells in te mature cochlea in vivo. Moreover, we have established RNA profiles of newly regenerated hair cells that identify transcription factors and signaling molecules that are likely involved in regulation of Atoh1-mediated hair cell regeneration in the mature cochlea. These novel and exciting findings led us to test whether combined genetic manipulation of these molecules would increase the efficiency of conversion of supporting cells to hair cells in the mature cochlea in vivo and determine the molecular mechanisms of such conversion. Direct conversion of supporting cells to hair cells is a key initial step toward hair cell regeneration and functional restoration of hearing. Our approach has been highly fruitful in generating new hair cells in vivo?both previously, in the postnatal cochlea, and now in the mature cochlea. For example, we have achieved a conversion rate of supporting cells to hair cells as high as 20% in vivo, a rate comparable to those in other regenerative systems such as adult pancreas, heart, and CNS. Our proposed studies will reveal 1) novel mechanisms of hair cell regeneration in the mature mammalian cochlea and 2) novel therapeutic targets for future clinical restoration of hearing.